Lactoferrin and transferrin are iron-binding proteins found in essentially all human secretions. Each of the proteins has a variety of properties, and both appear to contribute to antimicrobial host defense. In particular, each has been shown to have direct effects on pathogenic microorganisms including bacteriostasis and the induction of microbial iron uptake systems. It has been suggested that their primary antimicrobial mechanism is iron sequestration causing nutritional deprivation in susceptible organisms. However, a number of studies have indicated additional antimicrobial effects, including synergistic interactions with immunoglobins and complement, and a possible direct microbicidal effect for lactoferrin. In recent work we have found that lactoferrin and transferrin directly damage the outer membrane of Gram-negative bacteria. The proteins release LPS molecules from Gram-negative bacteria to differing degrees and enhance bacterial susceptibility to antibiotics, deoxycholate and lysozyme. The outer membrane effect of lactoferrin is dependent on direct cell contact, and lactoferrin can be shown to directly bind LPS. A peptide fragment of bovine lactoferrin has been identified that not only retains these outer membrane effects, but is also directly bactericidal. These effects on Gram-negative bacteria may be important properties of the proteins, and suggest a mechanism for synergy between the iron binding proteins and other host defense systems both systemically and in mucosal fluids such as salivary and gingival secretions. Our central hypothesis is that lactoferrin and transferrin can directly damage bacterial cells. Our aim is to precisely characterize how lactoferrin and transferrin mediate this bacterial cell damage using several complementary approaches. This information should provide insight into the overall role of the proteins in antimicrobial host defense. We first intend to characterize the structural elements of the lactoferrin molecule that mediate bacterial injury. In collaborative efforts we will use expression systems for bovine and human lactoferrin and site specific mutagenesis to test the role of specific molecular domains in antimicrobial activity. Second, we will address the effects of antibacterial peptide fragments of the proteins. Through the selection of resistant and sensitive mutant strains we will define the genetic basis for bacterial susceptibility to the effects of the antibacterial peptide fragments. Third, we will define whether the effect of lactoferrin and transferrin on Gram-negative cells enhances bacterial susceptibility to other host defense systems including the complement system and neutrophils.